Fluorescence Detection of Protein
Capillary gel electrophoresis using sodium dodecyl sulfate (CE-SDS) is used commercially to provide quantitative purity data for therapeutic protein characterization and release.
BiOptic, Inc. has developed and launched a new multi-channel capillary gel electrophoresis system that utilizes a multi-capillary gel-cartridge with a new polymer formulation and standardized methodology for enhanced detection of fluorescently labeled proteins. With the new Qsep400 system, we provide quality-controlled assay to the biotechnology industry, with specifications developed to assess the purity and heterogeneity of monoclonal IgG under either reduced or non-reduced states.
Introduction:
Electrophoresis is an analytical method frequently used in nucleic acid and protein analysis to separate macromolecules such as DNA, RNA, and proteins based on their size and charge. This technology is extensively used in the fields of medical diagnostics, life sciences, and clinical research.
The capillary electrophoresis system is an instrument used in the separation of nucleic acids and proteins with high accuracy and within a short span of time. Capillary electrophoresis is based on the principle of separation of molecules according to charge and size. The basic difference between capillary electrophoresis and gel electrophoresis is that the molecules are separated inside the small capillary tube which is filled with conductive buffer rather than gel. The separation of molecules using capillary electrophoresis is faster and gives higher resolution as the thin tubes have a higher surface-to-volume ratio, which enables them to dissipate heat faster and thus run at high voltages without overheating. Capillary electrophoresis requires a small amount of sample which is advantageous in the analysis of rare and expensive samples.
Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been used for size-based separations of proteins for over four decades, and it is still the workhorse for protein separations and analyses in most biological research laboratories.
The basic procedures of this method include: 1) preparing a gel and assembling the gel apparatus, 2) mixing a protein sample with a buffer containing SDS and cooking the mixture at an elevated temperature, 3) loading the protein-SDS mixture into the gel inside the gel apparatus and performing the electrophoresis, and 4) fixing, staining/de-staining, and quantitating the separated proteins. If SDS is allowed to react with sample proteins completely, the reactions should produce SDS-protein complexes having similar charge densities (or mass-to-charge ratios). When these complexes are electrophoretically separated, their mobilities will depend on their sizes, with smaller proteins having greater mobilities. The mobility values decrease linearly with the logarithm of protein molecular masses, which is the basic separation principle of SDS-PAGE. However, the technique is time-consuming and labor-intensive. The many manual operations (e.g., gel preparation, sample loading, staining/de-staining, etc.) are believed to be sources of SDS-PAGE irreproducibility.
SDS-capillary gel electrophoresis (SDS-CGE), also called capillary sieving electrophoresis (CSE) or capillary gel electrophoresis (CGE) shows many advantages over classical SDS-PAGE. These advantages include on-column detection, automated operation, great resolving power, and the capability of accurate protein quantification and molecular weight determination.
Methodology / Instrumentation:
The basic apparatus for CGE is identical to that of capillary zone electrophoresis (CZE) and consists of a capillary column, an on-column detector, and a high voltage power supply. The major difference between the two techniques is the separation media: a sieving matrix is employed in CGE while a background electrolyte solution is utilized in CZE.
The Qsep400TM is a simple-to-use CGE system that integrates micro-capillary electrophoretic technology into a plug-n-play disposable pen-shaped multi-capillary gel cartridge (Figure 1) with automated liquid handling (96-Samples), real-time fluorescent detection, and analysis.
Optimized for high resolution and high throughput at ambient temperatures, Qsep400 provides improved peak resolution, good linear dynamic range, and reproducible migration times with a lowered instrument and sample analysis cost.
Figure 1. The Qsep400 capillary gel electrophoresis Instrument with 4-channel gel-cartridge
Operation Steps of Qsep400:
1. Insert P2 gel-cartridge and place the samples
2. Select the proper method
3. Click "Run"
4. Analyze results
Materials and Methods:
Instrument Detection: Qsep400 (Ex/Em: 465-485 nm / 510-535 nm)
Cartridge: P2 gel-cartridge
Sample treatment: Sample dissolved in 1X Dilution Buffer and heat at 100°C for 5 minutes.
Method: Sample injection 4 KV for 5second, separation at 4 KV
CGE Analysis:
To evaluate the suitability of the system, the Protein Standard and unknown protein samples were separated by the multi-channel cartridge. Samples were electrokinetically introduced at 4 KV for 5 seconds and electrophoresis was performed at a constant voltage of 4 KV (Method: M-4-5-4kv).
A protein size ladder (BenchMarkTM) containing recombinant proteins of 11, 21, 32, 40, 63, 98, and 155 kDa was used to assist in the gel formulation phase to optimize the resolving capacity of the gel and to estimate the molecular weight (MW) of unknowns (Figure 2). In addition, the platform was adopted for evaluating the purity and heterogeneity of IgGs by 10 minutes (Figure 3), and also analyzed the composition of IgG, heavy chain, and light chain.
With ready to use gel cartridge, users can set up the instrument in 1 minute, and get results within 3-7 minutes (96 -samples in ~1.5 hours). The data can also be released by batch or individually with fully detailed information.
Figure 2. Protein Standard separation and molecular weight (MW) determination of an unknown sample by the multi-channel cartridge. Samples were electrokinetically introduced at 4KV for 5 seconds and electrophoresis was performed at a constant voltage of 4KV.
Figure 3. Biochemical characterization of sheep immunoglobulins (IgGs). Samples labeled with Chromeo P503 were treated with or without a reducing agent. Samples were electrokinetically introduced at 4 KV for 10 seconds and separated at a constant voltage of 4 KV.